Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: Correlation with the crystal structure

The crystal structure of the flavoprotein NADH peroxidase shows that the Ar g303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite ti tration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-elect ron reduced intermediate (EH2) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-S H is increased by over 3.5 units in comparison with the wildtype EH2 pK(a) of less than or equal to 4.5. NADH titration of the mutant peroxidase yield s the same EH2 intermediate, but in contrast to the behavior of wild-type e nzyme, this species can be reduced directly to an EH(2)(.)NAD(+) complex. K inetic analyses demonstrate that the R303M mutant is severely compromised, although active, with k(cat) = 3 s(-1) at pH 7.0, 5 degrees C; enzyme-monit ored turnover results indicate that the steady-state consists predominantly of an E-FADH(2)(.)NAD(+) species. When the oxidized mutant is reacted anae robically with 0.9 equiv of NADH/FAD, a clearly biphasic pattern is observe d at 450 nm; relatively rapid flavin reduction is followed by reoxidation a t 2.6-2.7 s(-1) (similar to k(cat)). Thus replacement of Arg303 with Met le ads to an altered peroxidase form in which the rate-limiting step in turnov er is the intramolecular transfer of electrons from FADH(2) --> Cys42-SOH. The crystal structure of the R303M peroxidase has been refined at 2.45 Angs trom resolution. In addition to eliminating the Arg303 interactions with Hi s10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein peroxidase.